Appendix H: Prion Research/Creutzfeldt-Jacob Disease (CJD) Guidelines

Creutzfeldt-Jacob Disease (CJD) is one of a group of neurodegenerative diseases called transmissible spongiform encephalopathies (TSE) (or prion diseases) which affect humans (e.g., Kuru, Fatal Familial Insomnia and Gerstmann-Sträussler-Scheinker syndrome of humans) and a variety of domestic and wild animal species (e.g., Scrapie of sheep, “Bovine Spongiform Encephalopathy [BSE] or Mad Cow Disease” of cattle and dairy cows, Chronic Wasting Disease [CWD] of deer and moose). Long (months to years) incubation periods precede the onset of clinical illness, with chronic progressive pathology that may last weeks to months. CJD is characterized by progressive dementia, myoclonic fasciculations, ataxia, and somnolence. The clinical course of CJD usually lasts several months and is invariably fatal. No effective treatment is available, and there are no known cases of remissions or recoveries.

A central biochemical feature of prion disease is the conversion of normal cellular prion protein (PrP) to an abnormal, misfolded, pathogenic isoform PrPSc (named for “scrapie”, the prototypic prion disease). The infectious agents that transmit prion disease are resistant to inactivation by heat and chemicals and thus require special biosafety precautions. Procedures involving brain tissue from patients with neurological degenerative disorders (such as CJD and Alzheimer’s disease) pose special challenges in reducing potential exposure to prions. Such material should be handled with at least the same precautions as HIV-positive or HBV-positive human tissue and prion-specific disinfection methods are recommended.

Laboratory Safety and Containment Recommendations

The CDC classifies prions as Risk Group 2 agents requiring Biosafety Level 2 (BSL2) containment. All researchers working with these agents are required to have IBC approval before any research can be initiated. In the laboratory setting prions from human tissue and human prions propagated in animals should be manipulated at BSL2. BSE (Bovine Spongiform encephalopathy) prions can likewise be manipulated at BSL2. However, due to the recent history of transmission of BSE prion to humans, work involving direct contact with BSE-infected specimens may require the use of BSL3 facilities and practices. All other animal prions are manipulated at BSL2.

This appendix mainly covers the lab safety and practice for research with prions that has evolved from best practices used at many institutions, including the University of California, San Francisco and the University of California, San Diego, that have been engaged in such research for years and also from the BMBL 6th edition Section VIII-H: Prion. Precautions for clinical safety and/or surgical procedures on patients diagnosed with prion diseases are not described here but are outlined in an Infection Control Guideline for TSE developed by a consultation convened by the WHO in 1999.

Safety Procedures

Prion agent must be treated as biohazardous. An autoclave for treatment of solid wastes is required. All equipment that has come in contact with the agent, packaging, containers and all unused portions and derivatives from the agent will be treated before disposal.

All fixed, non-fixed, or frozen tissues that contain the agent must be placed within watertight containers and labeled with the universal biohazard symbol and the notation “Infectious Materials.” The use of conventional autoclaving protocol as the sole treatment has not resulted in complete inactivation of prions. The recommended autoclaving procedure is in table below. Formalin-fixed and paraffin-embedded tissues, especially of the brain, remain infectious.

Personnel working with the agent must not have contact with any animal colonies without IACUC approval in the laboratory complex or with susceptible animal species.

Personnel must wear gloves and gowns while handling tissues that are potentially contaminated. All protective clothing must be removed before leaving the laboratory. Eye protection is recommended depending on the procedure. Personnel working with the material are instructed on the procedures for handling the agent. Sonication or homogenization of tissues must be performed in a properly certified Class II Biological Safety Cabinet (BSC).

The main precaution to be taken by laboratory members working with prion-infected or contaminated material is to avoid accidental puncture of the skin. Persons handling contaminated specimens should wear cut-resistant gloves if possible. If accidental contamination of unbroken skin occurs, the area should be washed with detergent and abundant quantities of warm water (avoid scrubbing); brief exposure (1 minute) to 1N NaOH or a 1:10 dilution of bleach may be considered for maximum safety.

The PI must contact the BSO in writing regarding spills and accidents that result in overt exposure to tissues. The report must include the following:

• Specification of amount released, time involved, and explanation of procedures used to determine the amount involved.
• Description of the area involved and the extent of employee exposure.
• Report of medical treatment provided.
• Corrective action taken to prevent the reoccurrence of the incident.

Records

ROHP must maintain health records for a period of 30 years. Records must be provided upon request by representatives of the Chief and/or Director of NIOSH. Access to the laboratory must be restricted to trained personnel when work is being conducted on tissue.

Personnel handling tissue must be trained in the following:

• Nature of CJD
• Route of transmission of CJD
• Specific hazards associated with handling of the tissue

Inactivation of Prions

Table 1. Prion Inactivation Methods for Reusable Instruments and Surfaces [BMBL 6th edition Section VIII-H: Prion Disease Table 4]

1. Immerse in 1 N NaOH or sodium hypochlorite (20,000 ppm available chlorine) for 1 hour. Transfer into water and autoclave (gravity displacement) at 121ºC for 1 hour. Clean and sterilize by conventional means. [Note: Sodium hypochlorite may be corrosive to some instruments, including autoclaves.]
2. . Immerse in a pan containing 1 N NaOH, heat in a gravity displacement autoclave at 121ºC for 30 minutes. Clean-rinse in water and sterilize by conventional means.
3. Immerse in 1 N NaOH or sodium hypochlorite (20,000 ppm) for 1 hour. Remove and rinse instruments with water, transfer to open pan and autoclave at 121ºC (gravity displacement) or 134ºC (porous load) for 1 hour. Clean and sterilize by conventional means.
4. Surfaces or heat-sensitive instruments can be treated with 2 N NaOH or sodium hypochlorite (20,000 ppm) for 1 hour. Ensure surfaces remain wet for entire period, then rinse well with water. Before chemical treatment, it is strongly recommended that gross contamination of surfaces be reduced because the presence of excess organic material will reduce the strength of either NaOH or sodium hypochlorite solutions.
5. 2% Environ LpH® (EPA Reg. No. 1043-118; no longer commercially available) may be used on washable, hard, non-porous surfaces (such as floors, tables, equipment, and counters), items, such as non-disposable instruments, sharps, and sharp containers, and/or laboratory waste solutions (such as formalin or other liquids). This product is currently being used under FIFRA Section 18 exemptions in a number of states. Users should consult with the state environmental protection office prior to use. Items may be immersed for 0.5–16 h, rinsed with water, and sterilized using conventional methods.

Working Solutions 1 N NaOH equals 40 grams of NaOH per liter of water. Solution should be prepared daily. A stock solution of 10 N NaOH can be prepared and fresh 1:10 dilutions (1 part 10 N NaOH plus 9 parts water) should be prepared frequently enough to maintain a fully effective alkalinity.

20,000 ppm sodium hypochlorite equals a 2% solution. Many commercial household bleach sources in the United States contain 6.15% sodium hypochlorite; for such sources, a 1:3 v/v dilution (1 part bleach plus 2 parts water) would produce a solution with 20,500 ppm available chlorine. This relatively easy method provides a slightly more concentrated solution (extra 500 ppm) that should not pose a problem with decontamination procedures or significantly increase chemical risks in the laboratory. Bleach solutions can off-gas and working solutions should be prepared frequently enough to maintain adequate available chlorine levels.

CAUTION: Above solutions are corrosive and require suitable personal protective equipment and proper secondary containment. These strong corrosive solutions require careful disposal in accordance with local regulations. Sodium, hypochlorite and sodium hydroxide solutions may corrode autoclaves.

Precautions in using NaOH or sodium hypochlorite solutions in autoclaves: NaOH spills or gas may damage the autoclave if proper containers are not used. The use of containers with a rim and lid designed for condensation to collect and drip back into the pan is recommended. Aluminum should not be used. Persons who use this procedure should be cautious in handling hot NaOH solution (post-autoclave) and in avoiding potential exposure to gaseous NaOH; exercise caution during all sterilization steps; and allow the autoclave, instruments, and solutions to cool down before removal. Immersion in sodium hypochlorite bleach can cause severe damage to some instruments. Neutral-ization of hypochlorite with thiosulfate prior to autoclaving is recommended to prevent the release of chlorine gas.